Dimerization-dependent serine protease activity of FAM111A prevents replication fork stalling at topoisomerase 1 cleavage complexes

FAM111A, a serine protease, plays roles in DNA replication and antiviral defense. Missense mutations in the catalytic domain cause hyper-autocleavage and are associated with genetic disorders with developmental defects. Despite the enzyme’s biological significance, the molecular architecture of the FAM111A serine protease domain (SPD) is unknown. Here, we show that FAM111A is a dimerization-dependent protease containing a narrow, recessed active site that cleaves substrates with a chymotrypsin-like specificity. X-ray crystal structures and mutagenesis studies reveal that FAM111A dimerizes via the N-terminal helix within the SPD. This dimerization induces an activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions. Dimerization is essential for proteolytic activity in vitro and for facilitating DNA replication at DNA-protein crosslink obstacles in cells, while it is dispensable for autocleavage. These findings underscore the role of dimerization in FAM111A’s function and highlight the distinction in its dimerization dependency between substrate cleavage and autocleavage.

Reviewer #1 (Remarks to the Author): In this manuscript, Machida and co-workers claimed to study the effect of dimerization on FAM111A serine protease activity and also on its allosteric property and biological functions such as antiviral activity and DNA replication using biochemical and structural biology tools.
The comments are as follows: The title 'FAM111A is a dimerization-dependent serine protease' is a very non-descript one that does not bring out the essence of the work and needs revision.The manuscript studies the role of dimerization of SPD of FAM111A on its activity and its associated biological functions.To look into this, they cloned SPD (a part of the full-length protein) and a few of its mutants and performed in vitro protease activity as well as studied their oligomeric properties.They also looked into the crystal structure of the SPD and a smaller mini SPD domain.
However, there are serious concerns regarding the experimental designs and subsequent interpretation of data: 1. SPD is a part of the full-length protein, therefore the oligomeric property and stability related to its oligomerization might not be relevant in the biological context.For example, one mutant they thought would make it a monomer, still showed dimeric property-and the reason provided that the concentration is high, is not clear to me.

2.
Oligomeric property was determined using SEC and AUC (sedimentation velocity).However, the oligomeric property is best determined through sedimentation equilibrium studies.Moreover, no Kd (dimerization constant) value has been provided that could have explained the strength of the dimers in all the protein variants and could have unambiguously explained the reason for the dimeric property observed in the mutant.Therefore, the data provided is very qualitative, not quantitative as expected from AUC.Since the entire manuscript depends on the oligomeric property, it makes no sense to not do so as it weakens the data provided and the interpretations made.
3. Many mutants were made that they predicted to make the SPD monomer-however, no rationale, however, has been provided on how those residues were chosen other than just structure-based wild guesses.An in silico analysis would have been a more organized approach.
4. X-ray crystallography was performed using a very high concentration of SPD domain that showed it is a dimer.Moreover, the monomer also showcased dimeric property and hence they made a truncated version of SPD (mini SPD) to determine its oligomeric property.Again, the entire experimental design is quite questionable.A dimer might be formed due to contacts at different parts of the protein, therefore chopping off a major part of the protein and concluding it is a monomer makes no sense; neither it proves anything nor it represents what actually happens in the cellular milieu.5. 'The differences observed between the two chains of the mini-SPD structure and the dimeric SPD as well as the higher B-factors observed for residues 536-538 encompassing the oxyanion hole and S1 pocket indicate that the monomeric SPD is more disordered.This suggests that dimerization of the SPD is associated with a disorder-to-order transition that stabilizes the oxyanion hole residues in the conformation that is competent for catalysis' This particular interpretation is an overstatement and is not backed by sufficient data.In fact, there is hardly any solid data to support it.Although some enzyme studies have been done, no data analysis using the Michaelis Menten equation has been made and no MM parameters have been provided that would show an allosteric property (Hill coefficient, Vmax, Km, etc).I am not sure on what basis the authors would say that the oxyanion hole is disrupted.Crystallography should have been backed with enzyme kinetics data.6. 'This might suggest that Tyr414 plays an additional role in the activation mechanism, possibly by transmitting the allosteric changes to other parts of the SPD such as the loop L6' Again, this statement is not backed by any data confirming the same.Allostery is a property that has not been proven anywhere in the manuscript; moreover, the fulllength protein might have provided a more comprehensive picture of the same.Therefore, cellbased studies would have been helpful since full length protein could not be purified in the bacterial system.
In the discussion, the authors talk about making allosteric inhibitors, which is more like talking in the air with no solid basis whatsoever.7. SPD R569H and D528G measured using Suc-AAP.However, why the activity of monomeric-= R569H is higher has not been explained.It was very much needed since it is counterintuitive and brings to question what is actually tried to be conveyed by this work.8. 'Expression of WT FAM111A prevented the spontaneous formation of TOP1cc foci in FAM111A KO, as described previously.In contrast, the expression of the monomeric mutants failed to prevent TOP1cc accumulation (Fig. 6b,c), despite their comparable expression levels to that of WT (Fig. 6a)' These are full-length FAM1111A protein variants used in cell-based assays and not the SPD whose oligomeric property was tested in vitro.Therefore, how it has been confirmed that the full-length mutants are also monomeric?9. How Functional assay correlates with in vitro protease activity?Since FAM111A localizes at replication forks and promotes DNA replication at protein obstacles through its protease activitythis could have been tested using appropriate cell-based assays to directly link protease activity with cellular functions.OVERALL: The work is very qualitative with data incomplete and hence it fails to prove the point addressed in the manuscript.

Reviewer #2 (Remarks to the Author):
In this study, the authors investigate the molecular architecture of the serine protease FAM111A, which is important for DNA replication in the presence of covalent DNA-protein crosslinks (DPCs).Missense germline mutations of FAM111A residues are also associated with genetic developmental disorders.The authors propose that FAM111A is a dimerization-dependent protease.X-ray crystal structures reveal that the SPD dimerizes via an N-terminal helix, inducing an allosteric activation cascade from the dimerization sensor loop to the oxyanion hole through disorder-to-order transitions.Replacement of key residues in FAM111A's dimerization interface results in loss or reduction of protease activity and protein stability in vitro, suggesting that enzyme oligomerization is important for FAM111A function.Moreover, dimerization-defective FAM111A variants fail to promote replication at DPC obstacles in cells.
Overall, these discoveries provide important information about FAM111A regulation.The manuscript is well-written, and the experiments support the conclusions.The following comments to the authors could guide some modest revisions.
1.The authors propose dimerization as a crucial regulatory mechanism that safeguards against uncontrolled FAM111A proteolysis.This implies that dimerization may be regulated.In the present study, the authors only investigate the catalytic SPD domain but it would be important to test whether the full-length protein also dimerizes and whether dimerization is affected by autocatalytic cleavage.Along these lines, autocleavage of dimerization-defective FAM111A V347D and V351D variants should be assessed in cells, as the authors have done in previous work.Could the increased expression levels of these variants in cells be related to defective autocleavage?FAM111A disease variants are generally believed to cause hyperactive protease activity.Interestingly, one mutation tested in the manuscript in context of the catalytic SPD domain decreased while another one increased activity.This may suggest that different patient mutations can have different effects on autocleavage than on substrate cleavage.Thus, it would be worthwhile to test whether the reported increased autocleavage of patient variants in cells requires dimerization of the SPD domain or not.2. The miniSPD and dimer-interface mutant variants are not only defective in dimerization and activity but are also less stable in vitro.While this suggests that dimerization is important for FAM111A, a structural biology expert should comment on whether some of the conclusions on allosteric activation of the active site are entirely justified or could be biased by unspecific folding defects.3. The authors conclude "..that the FAM111A SPD active-site architecture is tailored to cleave smaller substrates, such as linker regions or disordered substrates, and not globular proteins."However, it is also conceivable that FAM111A substrates are actively unfolded prior to proteolysis, as recently shown for SPRTN substrates.4. Figure 3C: The colors used are difficult to distinguish.

Reviewer #3 (Remarks to the Author):
FAM111A is a protein-coding gene important for cell-cycle regulation, and nuclear localization.The C-terminal part of the protein shares homology with trypsin-like peptidases and it contains a PCNA-interacting peptide (PIP) box, that is necessary for its co-localization with proliferating cell nuclear antigen (PCNA).In this study by Palani et al., investigated the structural and functional basis of the C-terminal part of the FAM111A.They have reported the first crystal structure of the C-terminal FAM111A (However, Alphafold model of full-length FAM111A (AF-Q96PZ2-F1) is already solved at a high confidence level) as both dimeric (active) and monomeric (inactive) configurations.They find that this protein exists as a dimer in solution and is necessary for its function whereas monomeric protein did not show any activity.Site-directed mutagenesis confirms the important residues for protein dimerization.Their structural data is supported by in-vitro protease activity data as well as cellular functional data to show that dimeric FAM111A is critical for TOP1cc accumulation and replication fork stalling in cells.Thus, the manuscript follows a previous publication from the same group (Kojima et al., 2020) where they show the different direction of the FAM111A, a PCNA-interacting protein, which plays an important role in mitigating the effect of protein obstacles on replication forks.This is a well-designed study with a substantial amount of high-quality data.That FAM111A is exist as a dimer in solution and is required for its function are significant findings, providing novel insights into how serine proteases are structurally flexible in their function.Although the work appears rigorous and essentially descriptive, the structural aspects do not break any significant new ground.Thus, I feel this report is too preliminary for publication in Nature Communication.
The following comments are provided for improving the manuscript.
1. Authors claim that FAM111A has chymotrypsin-like protease activity.However, they have just shown that FAM111A could cleave phenylalanine (Phe/F) at the P1 position.In contrast, chymotrypsin cleaves after Phe/Tyr/Trp residues.How could it be valid for P1 substitution by Tyr/Y or Trp/W?FAM111A still be able to cleave the substrate?2. I would describe the percentage of amino acid sequence identity and RMSD difference between FAM111A SPD and chymotrypsin in the result section.This will give the readers some idea of the difference between FAM111A SPD and previously characterized chymotrypsin.3. The crystal structures of C-terminal FAM111A are solved by molecular replacements using the Alphafold model.However, the authors did not report how good the alpha fold model to their crystal structure.I would add TFZ values and LLG values resulting from molecular replacement run into the method section.This will give a better understanding to the reader.3a I would list all these key interactions in the figure or in a separate panel.It makes easier for the reader to follow the idea.6. Page 09-paragraph 1, last sentence: "…..two sets of 6 hydrogen bonds and three salt bridges…".I would suggest authors to list those interactions in a figure or list them in-text.7.An interesting point is that both SPD mutants, V351 and V347D/V351D still retained the dimer interface.It is unclear what other residues/interactions are driving this dimer formation.8. How similar is the structure of mini-SPD to wt-SPD?I would report the RMSD value and any noticeable local structural changes/loop movements here.Also, this can be compared with the Alphafold structure available and discuss any structural changes.9. Few missing abbreviations, please use the full term when you use it the first time.

The reported final
-SV40 and PCNA 10.Missing reference on page 4, paragraph 2, line 5: "but FAM111A is one of the few proteases in this family that mainly localizes in the nucleus [REF]" Rfree value for the Mini SPD crystal structure is significantly high for 1.85A data.It is important to acknowledge what drives this high R-free value. 5. Page 8, second paragraph-last sentence: Report refers to Figures 3a and 3b to show a network of salt bridges and H-bonds.I can't see such interactions shown in that figure.also, this follows by, Page 9, line 2: can't see T563P mutant shown in supplementary figure